A variety of service and collaborative projects in protein characterization have been or are being carried out with the Protein Micro-characterization Core Facility (PMCF) with approximately 6500 samples analyzed from approximately 50 scientists representing 26 principle investigators from 8 laboratory branches. One large effort is in support of the Protein Expression Core Facility (PECF) and Dr. Bob Petrovich. The Role of the PMCF is to confirm gene expression at the protein level prior to the PECF handing materials over to their users. Other unpublished projects that are still ongoing include: Identification of binding partners and sites of post-translational modifications (PTMs) on transcription factors Raja Jothi Identification of binding partners and sites of post-translational modifications (PTMs) on transcription complexes Karen Adelman Identification of proteins in the BAF complexes from a variety of tissue types and/or conditions - Trevor Archer Puf family members binding partners - Traci Hall Other projects that have been recently published, or have been submitted and/or accepted for publication include: A report that TOPO1 is polynitrosylated by nitric oxide derived species, most likely at cysteine residues C300, C504, C505, and C630, resulting in significant down regulation of the protein via ubiquitin/26S proteasome pathway. Importantly, this down-regulation of TOPO1 resulted in a significant resistance to camptothecin. This resistance to camptothecin following nitrosylation did not result in the loss of the activity as there were no significant differences in TOPO1-induced DNA cleavage in vitro or in cells. B. Sinha and R. Mason A project showing that, in the absence of hormone, the nuclear receptor TR&#946; forms a cytoplasmic complex with the p85 subunit of PI3K and the Src family tyrosine kinase, Lyn, which depends on two canonical phosphotyrosine motifs in the second zinc finger of TR&#946; that are not conserved in TR&#945;. We found that when hormone is added, TR&#946; dissociates and moves to the nucleus, and PIP3 production goes up rapidly. When the rapid signaling mechanism was blocked chronically throughout development in mice by a targeted point mutation in both alleles of THRB and the mutation significantly impaired maturation and plasticity of the Schaffer collateral synapses on CA1 pyramidal neurons in the postnatal hippocampus. Thus, phosphotyrosine-dependent association of TR&#946; with PI3K provides a potential mechanism for integrating regulation of development and metabolism by thyroid hormone and receptor tyrosine kinases. - D. Armstrong We identified by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and NEDD4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus and promote Glis3 polyubiquitination. However, only Itch significantly reduced Glis3 stability by enhancing its proteasomal degradation. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate insulin gene expression and could provide a potential therapeutic target for Glis3-associated diseases, such as diabetes. A. Jetten We show PPIP5K2 has a conserved, polyarginine nuclear localization sequence (NLS), the function of which is supervised by Ser1006 phosphorylation. Mutation of either the NLS or Ser1006 alters the size of the nuclear pool of PPIP5K2 and impairs its ability to promote a pro-inflammatory activation of interferon-&#946; promoter activity. This demonstration of a mechanism to regulate PPIP5K2 function offers new directions for understanding nuclear PP-InsP activities. S. Shears Peanut extract was characterized by mass spectrometry (MS)and specific AGE modifications were found in raw and roasted peanuts and on rAra h 1 that was artificially gylcated by incubation with glucose or xylose. The binding of the RAGE-V1C1 domain to peanut allergens was assessed by PAGE and Western blotting with anti-Ara h 1, 2, and 3 antibodies. IgE binding to rAra h1 was assessed by PAGE and Western Blotting and RAGE was demonstrated to selectively interact with AGE modified rAra h 1. If the suggestion that sensitization to peanut allergens occurs in dendritic cells via RAGE is correct, these cells are likely interacting with modified Ara h 1, and Ara h 3, and not Ara h 2. G. Mueller and R. London We characterized some biochemical characteristics of the HeT-A Gag protein encoded by the HeT-A element of Drosophila. The HeT-A Gag protein when overexpressed in S2 cells was localized to the nucleus but was resistant to high salt, detergents and nuclease extraction treatments. Analysis of the HeT-A Gag protein by tandem mass spectrophotometry revealed that serines 216 and 221 are phosphorylated. Substituting these serines with alanine or aspartic acid by site-directed mutagenesis did not result in any changes in HeT-A Gag translocation across the nucleus, suggesting that phosphorylation of these sites is not associated with HeT-A Gag translocation, but time course experiments showed that these phosphorylation sites are important for Gag-protein stability. Additional projects that have required more than negligible resources include efforts performed with the Hu, Wilson, and R.S. Williams laboratories.